Conformation-specific anti-von Willebrand factor antibodies

Abstract

The present invention is directed to antibodies and antibody fragments that bind specifically to the active conformation of human von Willebrand factor. Most preferred are recombinantly produced single chain variable immunoglobulin fragments. Because the antibodies or antibody fragments act only at the sites of thrombus formation and do not interfere with the normal activity of circulating platelets, they are particularly well suited for use as antithrombotic agents in a wide variety of applications.

What is claimed is:  
     
         1 . A composition comprising an antibody that binds selectively to the active conformation of human von Willebrand Factor (vWF) and wherein said antibody inhibits the binding of vWF to platelets.  
     
     
         2 . The composition of  claim 1 , wherein said antibody is a single chain variable region immunoglobulin (ScFv) fragment.  
     
     
         3 . The composition of  claim 2 , wherein said ScFv fragment is from the mouse.  
     
     
         4 . The composition of any one of claims  1 - 3 , further comprising a pharmaceutically acceptable carrier.  
     
     
         5 . A method of identifying a single chain variable region immunoglobulin (ScFv) fragment that binds selectively to the active conformation of vWF, comprising: 
 a) immunizing an animal with an immunogen derived from the A1 region of human vWF;    b) isolating mRNA from the immunized animal of step a);    c) producing a cDNA library from the isolated mRNA of step b) in a bacteriophage capable of displaying ScFv fragments encoded by the cloned cDNA; and    d) screening the library of step c) to identify a bacteriophage displaying a ScFv fragment that binds selectively to the active conformation of vWF.    
     
     
         6 . The method of making and ScFv fragment that binds selectively to the active conformation of vWF, comprising: 
 a) immunizing an animal with an immunogen derived from the A1 region of human vWF;    b) isolating mRNA from the immunized animal of step a);    c) producing a cDNA library from the isolated mRNA of step b) in a bacteriophage capable of displaying ScFv fragments encoded by the cloned cDNA; and    d) screening the library of step c) to identify a bacteriophage displaying a ScFv fragment that binds selectively to the active conformation of vWF.    e) subcloning the recombinant DNA encoding the ScFv fragment identified in step d) in an expression vector;    f) transforming a host cell capable of expressing recombinant ScFv with the expression vector of step e); and    g) purifying the recombinant ScFv produced by the host cell of step f).    
     
     
         7 . The method of either  claim 5  or  6 , wherein the animal of step a) is a mouse.  
     
     
         8 . The method of either  claim 5  or  6 , wherein the screening of step d) comprises determining whether said ScFv fragment binds to vWF in the presence and absence of an agent that induces vWF to assume an active conformation.  
     
     
         9 . The method of  claim 8 , wherein said agent is Ristocetin.  
     
     
         10 . The method of either  claim 5  or  6 , wherein the screening of step d) comprises determining whether said ScFv fragment inhibits the binding of vWF to platelets.  
     
     
         11 . An ScFv fragment produced by the method of either  claim 5  or  6 .  
     
     
         12 . A method of inhibiting the binding of human vWF to platelets, comprising contacting said human vWF with an antibody that binds selectively to the active conformation of vWF.  
     
     
         13 . The method of  claim 12 , wherein said antibody is a ScFv fragment.  
     
     
         14 . The method of  claim 13 , wherein said ScFv is from the mouse.  
     
     
         15 . A method of preventing thrombus formation in a patient, comprising administering a pharmaceutical composition comprising an antibody that binds selectively to the active conformation of human vWF and that thereby prevents the binding of vWF to platelets, wherein said pharmaceutical composition is administered at a dosage sufficient to inhibit said thrombus formation.  
     
     
         16 . The method of  claim 15 , wherein said antibody is a ScFv fragment.  
     
     
         17 . The method of  claim 16 , wherein said ScFv fragment is from the mouse.